The most popular method of preventing reproductive activity in domestic animals, including dogs, horses, sheep, cattle, goats and cats, is surgical ovariohysterectomy or castration.
This method suffers from the problem that it is irreversible and is, technically, a relatively difficult procedure, therefore requiring the skills of trained veterinarians.
One of the alternative methods to surgery is the administration of progestagen steroids which can be used as long term oestrus suppressants (Harris and Wotchuk Am. J. Vet. Res. 24: 1003-1006, 1963) in dogs, but are unfortunately associated with the induction of uterine disorders including pyometritis, endometritis and increased incidence of benign mammary tumours following long term treatment. Their use has therefore tended to become confined to short term suppression of oestrus or postponement of oestrus.
In economically important farm animals there is no commonly used long term contraceptive which has been found to be suitable for routine use in the field.
There is therefore a need for a well-tolerated non-steroidal method of contraception in domestic animals which is applicable to both male and female domestic animals.
One such method would be to immunise against the hormones which control the development and activity of the reproductive organs.
The two gonadotrophic hormones which regulate gonadal steroidogenesis and gametogenesis, and are responsible for reproductive cyclicity are luteinizing hormone (LH) and follicle stimulating hormone (FSH).
Luteinizing hormone releasing hormone (LHRH, also known as GnRH) controls the synthesis and release of LH and FSH from the anterior pituitary gland. Mammalian LHRH is a decapeptide comprised of naturally occurring amino acids in the following sequence (ID NO: 1): EQU (pyro)--Glu--His--Trp--Set--Tyr--Gly--Leu--Arg--Pro--Gly--NH.sub.2
The N and C terminal glutamic acid and glycine residues are modified after translation to pyroglutamic acid and glycinamide respectively. Vaccines which result in the production of antibodies against LHRH by a host will suppress that host's endogenous LH and FSH production and release. This suppression can result in reduction of steroidogenesis and a failure of reproductive cyclicity and fertility in the treated animal. The resultant physiological effects are
(a) in the female: PA0 (b) in the male:
(i) a cessation of LH pulsatility,
(ii) a failure of ovulation leading to infertility
(iii) a cessation of oestrus cycles due to the lack of oestrogens,
(iv) regression of the reproductive tract
(v) abortion due to regression of the corpus luteum
A suppression of production of testosterone from the Leydig cells in the testes resulting in lowered peripheral blood serum levels of circulating androgens, causes:
(i) reduced libido,
(ii) regression of the accessory sex glands, and
(iii) diminution in the testicular volume and reduction/cessation of spermatogenesis.
Antibodies against LHRH can be produced in a number of species by chemically conjugating LHRH to a suitable carrier and administering it in the presence of an appropriate adjuvant (Carrelli C. et al, 1982, Proc. Natl. Acad. Sci USA 79 5392-5395). Chemical conjugation is however, difficult to control and often results in a heterogeneous and ill-defined product. Moreover, an oil-based adjuvant is usually required for effective immunisation and this often leads to the formation of unacceptable side effects such as inflammation and granulomatous tissue lesions.
It is desirable to provide a means for producing good titres of antibodies against LHRH without the need to use strong adjuvants.
The TraT protein (TraTp) is coded by the TraT gene. TraTp is an outer membrane lipo-protein produced by certain strains of E. coli and is responsible for the resistance of these strains to killing by serum. When injected intramuscularly into mice, without adjuvant, TraTp elicits an antibody response which is comparable to that obtained when it is injected with incomplete Freund'sadjuvant. Furthermore, chemical coupling of an immunogen to TraTp followed by administration of the complex in saline to an animal results in the production of high levels of anti-immunogen antibodies. TraTp, therefore, can be used as a self-adjuvanting carrier of immunogens. This use of TraTp has been described previously in International Patent Application No. PCT/AU87/00107 (published as WO 87/06590), wherein both chemical and genetic linkage of TraTp to immunogen molecules was described. The specific fusions made and described in that specification relate to large proteins. On the other hand, LHRH is a short peptide which makes it inherently difficult to use as an immunogen without a suitable carrier. Furthermore, as there is little variation in the peptide between species, it is seen as a self-antigen by the immune system and is consequently recalcitrant to the stimulation of an immune response. Fusion proteins comprising LHRH sequences and LTB (the B subunit of the heat labile toxin produced by certain strains of E. coli) have been described (International Patent Application No. PCT/AU86/00135 published as WO86/06635). These constructs were prepared for the purpose of orally presenting LHRH to the immune system of a host, using the ability of LTB to bind to mucosal epithelium. They are not self-adjuvanting and although inhibition of reproductive function was demonstrated, the resulting inhibition was not a strong inhibition.
PCT/EP89/01013 (published as WO 90/02187) describes the production of fusion proteins including a peptide which alone is not substantially antigenic such as LHRH using a "carrier" which is a highly antigenic, hydrophilic protein such as hepatitis B surface antigen. TraTp is a membrane lipoprotein and is not a highly hydrophilic protein. Further the fusions taught in PCT/EP89/01013 do not appear to be self-adjuvanting.
______________________________________ Abbreviations ______________________________________ LHRH: Luteinizing Hormone Releasing Hormone LH: Luteinizing Hormone GnRH: Gonadotrophin Releasing Hormone (is another name for LHRH) FSH: Follicle Stimulating Hormone LTB: The B subunit of the heat-labile toxin produced by certain strains of E. coli QC: Quality Control QA: Quality Assurance EDTA: Ethylene diaminetetra-acetic acid SDS: Sodium Dodecyl Sulphate SDS-PAGE: Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis LPS: Lipopolysacharide EDAC: 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide HCl ABTS: 2,2.sup.1 -Azinobis(3-ethylbenzthiazoline sulphonic acid) PEG: Polyethylene glycol BSA: Bovine Serum Albumin IF: Insoluble form of the fusion protein SF: Soluble form of the fusion protein PHA: Phytohaemagglutinin MBS: m-maleimido benzoic acid n-hydroxysuccinimide ester A.sub.280 : Absorbance, at a setting of 280 nm on the spectrophotometer ISA-20: Montanide adjuvant, SEPPIC ISA-25: Montanide adjuvant, SEPPIC sem: Standard error of the Mean sd; Standard deviation PBS: Phosphate Buffered Saline, pH 7.2-7.4 w/w: Weight for weight v/w: Volume for weight DNA: Deoxyribonucleic acid NSB: Non-specific binding ______________________________________